EP0764048B1 - Chromatographische adsorbenden, die merkaptoheterozyklische liganden anwenden - Google Patents

Chromatographische adsorbenden, die merkaptoheterozyklische liganden anwenden Download PDF

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EP0764048B1
EP0764048B1 EP95919182A EP95919182A EP0764048B1 EP 0764048 B1 EP0764048 B1 EP 0764048B1 EP 95919182 A EP95919182 A EP 95919182A EP 95919182 A EP95919182 A EP 95919182A EP 0764048 B1 EP0764048 B1 EP 0764048B1
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ligand
solid support
support material
mercapto
chromatography adsorbent
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EP0764048A4 (de
EP0764048A1 (de
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Alexander Schwarz
Meir Department Of Biophysics Wilchek
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Aspira Womens Health Inc
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Biosepra Inc
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/3092—Packing of a container, e.g. packing a cartridge or column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
    • B01J2220/46—Materials comprising a mixture of inorganic and organic materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/58—Use in a single column

Definitions

  • Affinity chromatography which relies on specific interactions between an immobilized ligand and a particular molecule sought to be purified, is another well-known, existing technique used to purify antibodies from solution.
  • Protein A which is derived from the bacterium Staphylococcus aureus , has a strong and specific affinity for the Fc fragment of IgG antibodies and has been used for approximately one decade as an affinity ligand for purifying IgG antibodies.
  • Protein A can be immobilized on a large variety of solid support materials, such as chromatographic beads and membranes, and can be used to obtain high purity antibodies in high yield.
  • Protein A suffers from a number of limitations as an affinity ligand.
  • One such limitation is the prohibitive cost of Protein A. Because of its high cost, the large scale utilization of Protein A as an affinity ligand is not often economically feasible.
  • Another such limitation is the sensitivity of Protein A to those proteases that are commonly present in the same biological fluids (e.g., sera, ascitic fluids, milk, hybridoma cell culture supernatants, and the like) in which the antibodies being sought to be purified by affinity chromatography are also present.
  • Protein L from Peptostreptococcus magnus. Protein L, as contrasted with Protein A and Protein G, interacts specifically with the light chains of IgG antibodies without interfering with their antigen binding sites. This specificity permits Protein L to complex not only with antibodies of the IgG class but also with antibodies of the IgA and IgM classes. Despite its broad affinity, Protein L suffers from the same limitations described above in connection with Proteins A and G.
  • Proteins A, G and L are all sensitive to a number of chemical and physical agents (e.g., extreme pH, detergents, chaotropics, high temperature) which are frequently used to clean affinity chromatography columns between runs. Consequently, some people have chosen to minimize the number of cleaning cycles applied to the above-described columns so as to correspondingly minimize degradation thereto.
  • One drawback to this tactic is that failure to clean the columns regularly prevents optimal antibody purification.
  • Anti-antibodies represent still another type of affinity ligand used for gamma globulin purification. Anti-antibodies, however, are limited in use due to their high cost and very limited stability.
  • Thiophilic compounds represent another class of pseudobioaffinity ligands.
  • An adsorbent utilizing one type of thiophilic compound is disclosed by Porath et al. in FEBS Lett ., 185 :306 (1985). This type of adsorbent is produced by reacting either a hydroxyl- or thiol-containing support first with divinyl sulfone and then with mercaptoethanol. The aforementioned adsorbent utilizes a salt-promoted approach to adsorb immunoglobulins. Elution of adsorbed immunoglobulins is effected by decreasing salt concentration and/or by modifying pH.
  • pseudobioaffinity adsorbent capable of adsorbing antibodies utilizes mercaptopyridine as its ligand. See Oscarsson et al., "Protein Chromatography with Pyridine- and Alkyl-Thioether-Based Agarose Adsorbents," Journal of Chromatography , 499 :235-247 (1990).
  • This type of adsorbent is generated, for example, by reacting mercaptopyridine with a properly activated solid support. The adsorbent thus formed is capable of adsorbing antibodies under high salt conditions.
  • thiophilic adsorbents provide a generally satisfactory means for purifying antibodies; however, in those cases in which the initial biological liquid is a protein rich solution, such as a serum or ascites, the non-specific binding by such adsorbents of a number of proteins other than the desired antibodies can be a problem. This problem of non-specific binding is the primary limitation of these thiophilic adsorbents.
  • Another group of low molecular weight ligands capable of selectively binding antibodies includes pentafluoropyridine and N-dimethylaminopyridine reacted with ethylene glycol, glycine or mercaptoethanol. See Ngo, J . Chromatogr ., 510 :281 (1990).
  • Adsorbents utilizing these materials can be used to isolate immunoglobulins in either high salt or low salt buffers or to isolate other types of proteins under low salt conditions. Elution of adsorbed proteins can be obtained by lowering pH.
  • a pseudobioaffinity chromatography adsorbent adapted for use in selectively adsorbing immunoglobulins
  • the adsorbent comprising in a first embodiment: (a) a solid support material; and (b) a ligand immobilized on the surface of the solid support material, said ligand being a compound of the formula (I) of Claim 1.
  • the present invention is directed to novel pseudobioaffinity chromatography adsorbents useful in the selective adsorption of a wide of range of immunoglobulins.
  • the adsorbents of the present invention may also be used to selectively adsorb non-immunoglobulin proteins.
  • the pseudobioaffinity chromatography adsorbents comprise (a) a solid support material; and (b) a ligand immobilized on the surface of the solid support material, the ligand being a mercapto five-membered heterocyclic ring of the type hereinafter described.
  • said ligand has the structure represented by the formula (I) of Claim 1.
  • compound I examples include mercaptothiazoline (e.g., 2-mercaptothiazoline) and 2-mercapto-5-thiazolidone.
  • said ligand has the structure represented by the formula (II) of claim 4.
  • the solid support material of the present adsorbents may be composed of polysaccharides, such as cellulose, starch, dextran, agar or agarose, or hydrophilic synthetic polymers, such as substituted or unsubstituted polyacrylamides, polymethacrylamides, polyacrylates, polymethacrylates, polyvinyl hydrophilic polymers, polystyrene, polysulfone or the like.
  • suitable materials for use as the solid support material include porous mineral materials, such as silica, alumina, titania, zirconia and other ceramic structures.
  • composite materials may be used as the solid support material.
  • Such composite materials may be formed by the copolymerization of or by an interpenetrated network of two or more of the above mentioned entities.
  • suitable composite materials include polysaccharide-synthetic polymers and/or polysaccharide-mineral structures and/or synthetic polymer-mineral structures, such as are disclosed in U.S. Patent Nos. 5,268,097, 5,234,991 and 5,075,371.
  • the solid support material of the present invention may take the form of beads or irregular particles ranging in size from about 0.1 mm to 1000 mm in diameter, fibers (hollow or otherwise) of any size, membranes, flat surfaces ranging in thickness from about 0.1 mm to 1 mm thick and sponge-like materials with holes from a few ⁇ m to several mm in diameter.
  • the ligands described above are chemically immobilized on the solid support material via a covalent bond formed between the mercapto group of the ligand and a reactive group present on the solid support.
  • Reactive groups capable of reacting with the mercapto group of the present ligand include epoxy groups, tosylates, tresylates, halides and vinyl groups. Because many of the aforementioned solid support materials do not include one of the reactive groups recited above, bifunctional activating agents capable of both reacting with the solid support materials and providing the necessary reactive groups may be used.
  • the amount of immobilized ligand of the present invention can vary anywhere between a fraction of a ⁇ mole and several hundred ⁇ moles per ml of solid matrix.
  • a small quantity of ligand per unit volume results in a low separation capacity for immunoglobulins whereas a large quantity of ligand per unit volume results in an increased sorption capacity for immunoglobulins (and can induce nonspecific binding of proteins other than immunoglobulins).
  • the activating agents listed above have different chain lengths. Accordingly, by selecting a particular activating agent, one can control the distance between the solid support material and the immobilized ligand. Due to steric constraints, this distance between the ligand and the solid matrix can affect the adsorption characteristics of the final product both in terms of specificity and sorption capacity. Generally, more ligand can be immobilized on the solid support material in those instances in which the chain length is great than in those instances in which it is not.
  • the washed, activated support is then contacted with ligands of the type described above under established conditions of concentration, pH, reaction temperature, reaction medium, time and agitation to produce an adsorbent which selectively adsorbs immunoglobulins from biological liquids contacted therewith.
  • the adsorbents of the present invention may be used in the same manner that conventional adsorbents have been used. This comprises introducing the adsorbent into a chromatographic column and then washing the column with an appropriate aqueous solution of salts at a given pH. A biological solution containing the immunoglobulins wished to be isolated is then combined with salts (e.g., Na 2 SO 4 ) and directly loaded into the column. The immunoglobulins present in the solution are captured by the adsorbent whereas the non-immunoglobulin proteins present in the solution and the remainder of the solution are recovered in the flowthrough. After an appropriate washing to eliminate non-specifically adsorbed macromolecules, the immunoglobulins are then desorbed by a pH shift and/or by changing the salt concentration. The isolated immunoglobulins are then neutralized.
  • salts e.g., Na 2 SO 4
  • adsorbents of the present invention do not require that salt be added to the biological solution for the antibodies contained therein to adsorb to the ligand. This elimination of the need for salt is clearly advantageous in those instances in which the addition of salt is undesirable.
  • Those adsorbents of the present invention for which salt need not be added (and, in fact, cannot be added in order for the adsorbent to function properly) are those which employ ligands of compounds I and II in which nitrogen atoms constitute the two or more heteroatoms of the five-membered heterocyclic ring.
  • Examples of such ligands include 2-mercaptoimidazole, 3-mercapto-1,2,4-triazole, 5-mercapto-1,2,3-triazole, 5-mercapto-1,2,3,4-tetrazole and N-methyl-2-mercapto-imidazole.
  • the adsorbents of the present invention can be used to isolate a variety of different types of antibodies, such as native antibodies, chemically modified antibodies, bioengineered antibodies, antibody fragments and antibody conjugates containing enzymes, as well as various toxins, haptens and the like.
  • the adsorbents of the present invention can be used to isolate antibodies from a variety of different biological liquids, such as hybridoma cell culture supernatants, plasma and plasma fractions, milk and milk fractions, ascitic fluids, fermentation broths and the like.
  • the adsorbents of the present invention represent a significant advancement in the art over adsorbents utilizing antibody-specific proteins, such as Protein A, Protein G, Protein L and the like. This is, in part, because the present adsorbents are stable against biological degradation, particularly degradation by hydrolytic enzymes.
  • many of the ligands of the present adsorbents are commercially available chemicals of a current cost significantly lower than protein-based ligands.
  • the present adsorbents can be treated with strong acidic and alkaline solutions which would otherwise be inappropriate with adsorbents utilizing protein ligands.
  • the present adsorbents can be treated with other cleaning agents, such as detergents, chaotropic salts and the like, without running the risk of adsorbent degradation or inactivation.
  • heat treatment can be applied to the adsorbents of the present invention without modifying their capture efficiency or specificity.
  • the present adsorbents are more specific and/or show higher sorption capacity for antibodies.
  • Example 1 Immobilization of 2-mercaptoimidazole on an epoxy-activated HyperDTM support
  • the beads were then resuspended in 2 M sodium acetate, and 2 ml of acetic anhydride was added over a period of one hour during shaking. The mixture was then shaken for an additional hour, filtered onto a sinter glass filter and washed with water until neutrality.
  • a chromatographic assay was then performed on the above-described adsorbent using 10 mM HEPES at pH 7.5. 1.5 ml of human serum was diluted three times with HEPES buffer and passed through a column of 1.2 ml gel volume. Elution was performed by changing the pH to 3.5 with citric acid buffer. Electrophoretic analysis of the elution fractions collected revealed that immunoglobulins constituted the major part of the adsorbed proteins, and the purity of the immunoglobulins was judged to be greater than 75%.
  • the dynamic capacity of the modified gel by frontal analysis was 9.2 mg/ml at c/10 (i.e., 10% breakthrough) and 14.4 mg/ml at c/2 (i.e., 50% breakthrough) for human immunoglobulins at a flow rate of 120 cm/hr.
  • Example 2 An adsorbent similar to that synthesized in Example 1 was prepared, the only difference being that 2-aminoimidazole was substituted for 2-mercaptoimidazole.
  • An evaluation of the dynamic capacity of the adsorbent was performed using frontal analysis and showed that the 2-aminoimidazole adsorbent had a dynamic capacity of 1.5 mg/ml at c/10 and 2.5 mg/ml at c/2 for human immunoglobulins at a flow rate of 120 cm/hr.
  • Example 2 An adsorbent similar to that synthesized in Example 1 was prepared, the only difference being that 2-mercaptothiazole was substituted for 2-mercaptoimidazole.
  • a chromatographic assay was performed using 10 mM HEPES and 500 mM sodium sulfate at pH 7.5. 1.5 ml of human serum was passed through a column of 4.6 ml gel volume, and elution was performed by changing the pH to 3.5 with citric acid buffer.
  • Electrophoretic analysis of the elution fractions collected revealed that immunoglobulins constituted the major part of the adsorbed proteins, and the purity of the immunoglobulins was judged to be greater than 85%.
  • the capacity of the modified gel by frontal analysis was determined to be 6.2 mg/ml at c/10 and 7.9 mg/ml at c/2 at a flow rate of 120 cm/hr.
  • Example 2 An adsorbent similar to that synthesized in Example 1 was prepared, the only difference being that 2-mercaptothiazoline was substituted for 2-mercaptoimidazole.
  • a chromatographic assay was performed using 10 mM HEPES and 500 mM sodium sulfate at pH 7.5. 1.5 ml of human serum was passed through a column of 4.6 ml gel volume, and elution was performed by changing the pH to 3.5 with citric acid buffer.
  • Electrophoretic analysis of the elution fractions collected revealed that immunoglobulins constituted the major part of the adsorbed proteins, and the purity of the immunoglobulins was judged to be greater than 85%.
  • the capacity of the modified gel by frontal analysis was determined to be 6.2 mg/ml at c/10 and 7.7 mg/ml at c/2 at a flow rate of 120 cm/hr.
  • Example 5 lmmobilization of 3-mercapto-1,2,4-triazole on an epoxy-activated HyperDTM support
  • Example 2 An adsorbent similar to that synthesized in Example 1 was prepared, the only difference being that 3-mercapto-1,2,4-triazole was substituted for 2-mercaptoimidazole.
  • the capacity of the modified gel for pure human antibodies was determined by frontal analysis to be 1.5 mg/ml at c/10 and 8.1 mg/ml at c/2 at a flow rate of 146 cm/hr and an adsorption buffer containing 10 mM HEPES at pH 7.
  • Example 2 An adsorbent similar to that synthesized in Example 1 was prepared, the only difference being that 2-mercapto-1,3,4-thiadiazole was substituted for 2-mercaptoimidazole.
  • the capacity of the modified gel for pure human antibodies was determined by frontal analysis to be 9.1 mg/ml at c/10 and 11.2 mg/ml at c/2 at a flow rate of 140 cm/hr in an adsorption buffer containing 10 mM HEPES and 750 mM sodium sulfate at pH 7.
  • Example 2 An adsorbent similar to that synthesized in Example 1 was prepared, the only difference being that 2-mercapto-5-thiazolidone was substituted for 2-mercaptoimidazole.
  • the capacity of the modified gel for pure human antibodies was determined by frontal analysis to be 9 mg/ml at c/10 and 12.2 mg/ml at c/2 at a flow rate of 134 cm/hr in an adsorption buffer containing 10 mM HEPES and 750 mM sodium sulfate at pH 7.
  • Example 8 Immobilization of 2-mercaptothiazoline on a vinyl-activated HyperDTM support
  • the slurry was then filtered onto a sinter glass filter, washed with ethanol and dried.
  • the modified HyperDTM support was then resuspended in 100 ml of a solution containing 1 M ethanolamine, pH 10.5, shaken for 2.5 hours at 45°C, filtered and washed with water.
  • a chromatographic assay was performed using 10 mM HEPES and 500 mM sodium sulfate at pH 7.5. 1.5 ml of human serum was passed through a column of 4.6 ml gel volume, and elution was performed by changing the pH to 2.5 with citric acid buffer. Electrophoretic analysis of the elution fractions collected revealed that immunoglobulins constituted the major part of the adsorbed proteins, and the purity of the immunoglobulins was judged to be greater than 85%.
  • the capacity of the modified gel was determined by frontal analysis to be 11.8 mg/ml at c/10 and 14.4 mg/ml at c/2 at a flow rate of 120 cm/hr.
  • Example 9 Immobilization of 2-mercaptothiazole on a bromo-activated HyperDTM support
  • the modified gel was then evaluated with pure human IgG. Frontal analysis of the modified gel showed a dynamic capacity of 2.2 mg/ml at c/10 and 7.8 mg/ml at c/2 at a flow rate of 160 cm/hr using 10 mM HEPES and 500 mM Na 2 SO 4 at pH 7.0.
  • Epoxy-Sepharose CL-6B support epoxy-activated agarose beads with an epoxy content of 10-12 ⁇ M/ml of gel; commercially available from Pharmacia Biotech, Piscataway, NJ
  • Epoxy-activated agarose beads with an epoxy content of 10-12 ⁇ M/ml of gel commercially available from Pharmacia Biotech, Piscataway, NJ
  • the slurry was rotated, head over head, for 20 hours and then filtered onto a glass filter, washed and drained.
  • the agarose beads were then resuspended in an aqueous solution of 1 M ethanolamine at pH 10.5 and rotated, head over head, for two hours at room temperature.
  • the slurry was then filtered and washed with water until neutrality and then resuspended in 2 M sodium acetate. 1 ml of acetic anhydride was then added over the course of one hour. The slurry was then rotated for an additional hour, filtered onto a sinter glass filter, washed to neutrality and drained.
  • the modified gel was evaluated with pure human IgG. Frontal analysis of the modified gel showed a dynamic capacity of 5.4 mg/ml at c/10 and 24.2 mg/ml at c/2 at a flow rate of 75 cm/hr using 10 mM HEPES and 500 mM Na 2 SO 4 at pH 7.0.
  • Eupergit® support a copolymer of methacrylamide, N,N-methylenebis(methacrylamide) and a component containing an active oxirane group; commercially available from Rohm Pharma, Darmstadt, Germany
  • 50 ml of 50% 50 mM carbonate buffer, pH 8.6, and 50% ethanol To this suspension are added 50 ml of ethanol containing 5 mM of 2-mercapto-1,3,4-thiadiazole, and the slurry is shaken for 16 hours. The slurry is then filtered, washed with 50 mM carbonate buffer at pH 8.6/ethanol (1:1) and drained under vacuum.
  • the modified support is then saturated with ethanolamine as described in Example 1 and tested for its ability to separate antibodies from human plasma. Its sorption capacity is also checked by frontal analysis using pure human IgG and is expected to be within ⁇ 10% of that disclosed for the adsorbent in Example 6.
  • Epoxy-Fractogel support epoxy-activated polymethacrylate support; commercially available from E. Merck, Wakefield, RI
  • 50 ml of 50% of 50 mM carbonate buffer, pH 8.6, and 50% ethanol To this suspension are added 50 ml of ethanol containing 5 mM of 2-mercapto-1,3,4-thiadiazole, and the slurry is shaken for 16 hours. The slurry is then filtered, washed with 50 mM carbonate buffer at pH 8.6/ethanol (1:1) and drained under vacuum.
  • the modified support is then saturated with ethanolamine as described in Example 1 and tested for its ability to separate antibodies from human plasma. Its sorption capacity is also checked by frontal analysis using pure human IgG and is expected to be within ⁇ 10% of that disclosed for the adsorbent in Example 6.
  • Example 14 Purification of immunoglobulins from bovine colostrum on 2-mercaptoimidazole
  • Bovine colostrum was diluted 1:1 with adsorption buffer (10 mM HEPES, pH 7.3) and filtered. Adsorption was performed on a 5.9 ml column at a flow rate of 120 cm/hr. Elution was performed by 200 mM sodium dihydrogen phosphate, pH 4.25. Electrophoretic analysis revealed that the immunoglobulins eluted were greater than 80% pure.

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Claims (15)

  1. Pseudobioaffinitätschromatographie-Adsorptionsmittel zur Verwendung bei der selektiven Adsorption von Immunoglobulinen, wobei das Pseudobioaffinitätschromatographie-Adsorptionsmittel umfasst:
    (a) ein festes Trägermaterial; und
    (b) einen Liganden, der an der Oberfläche des festen Trägermaterials über eine kovalente Bindung immobilisiert ist, die zwischen der Mercaptogruppe des Liganden und einer auf dem festen Trägermaterial vorliegenden reaktiven Gruppe ausgebildet ist, wobei der Ligand die Formel I hat:
    Figure 00300001
       wobei jedes von X1, X2 und X3 aus S, SCH3 +, O, NH, NCH3, CH2 und CR1R2 ausgewählt ist, wobei R1 und R2 aus substituiertem oder unsubstituiertem Alkyl, Aryl, Alkenyl, Alkinyl, Aralkyl, Acyl, Cycloalkyl, Carboxyl, Amino, Aryloxy oder Alkoxy, Halogen, Hydroxy, Nitro, O, S und Cyano ausgewählt sind; wobei X4 aus N, NCH3 +, CH und CR ausgewählt ist, wobei R aus substituiertem oder unsubstituiertem Alkyl, Aryl, Alkenyl, Alkinyl, Aralkyl, Acyl, Cycloalkyl, Carboxyl, Amino, Aryloxy oder Alkoxy, Halogen, Hydroxy, Nitro, O, S und Cyano ausgewählt ist; und wobei wenigstens zwei von X1, X2, X3 und X4 weder CH2, CH, CR noch CR1R2 sind, und wobei die reaktive Gruppe aus Epoxygruppen, Tosylaten, Tresylaten, Halogeniden und Vinylgruppen ausgewählt ist.
  2. Pseudobioaffinitätschromatographie-Adsorptionsmittel nach Anspruch 1, wobei der Ligand aus Mercaptothiazolin ausgewählt ist, insbesondere 2-Mercaptothiazolin und 2-Mercapto-5-thiazolidon.
  3. Pseudobioaffinitätschromatographie-Adsorptionsmittel nach Anspruch 1, wobei keines von X1, X2 und X3 S, SCH3 + oder O ist.
  4. Pseudobioaffinitätschromatographie-Adsorptionsmittel zur Verwendung bei der selektiven Adsorption von Immunoglobulinen, wobei das Pseudobioaffinitätschromatographie-Adsorptionsmittel umfasst:
    (a) ein festes Trägermaterial; und
    (b) einen Liganden, der an der Oberfläche des festen Trägermaterials über eine kovalente Bindung immobilisiert ist, die zwischen der Mercaptogruppe des Liganden und einer auf dem festen Trägermaterial vorliegenden reaktiven Gruppe ausgebildet ist, wobei der Ligand die Formel II hat:
    Figure 00310001
       wobei Y1 aus S, SCH3 +, O, NH, NCH3, CH2 und CR1R2 ausgewählt ist, wobei R1 und R2 aus substituiertem oder unsubstituiertem Alkyl, Aryl, Alkenyl, Alkinyl, Aralkyl, Acyl, Cycloalkyl, Carboxyl, Amino, Aryloxy oder Alkoxy, Halogen, Hydroxy, Nitro, O, S und Cyano ausgewählt sind; wobei jedes von Y2, Y3 und Y4 aus N, NCH3 +, CH und CR ausgewählt ist, wobei R aus substituiertem oder unsubstituiertem Alkyl, Aryl, Alkenyl, Alkinyl, Aralkyl, Acyl, Cycloalkyl, Carboxyl, Amino, Aryloxy oder Alkoxy, Halogen, Hydroxy, Nitro, O, S und Cyano ausgewählt ist; und wobei wenigstens zwei von Y1, Y2, Y3 und Y4 weder CH2, CH, CR noch CR1R2 sind, und wobei die reaktive Gruppe aus Epoxygruppen, Tosylaten, Tresylaten, Halogeniden und Vinylgruppen ausgewählt ist.
  5. Pseudobioaffinitätschromatographie-Adsorptionsmittel nach Anspruch 4, wobei der Ligand aus Mercaptoimidazol, Mercaptoimidazolin, Mercaptothiazol, Mercaptotriazol, Mercaptotetrazol, Mercaptothiadiazol und Mercaptomethylimidazol ausgewählt ist; insbesondere wobei der Ligand aus N-Methyl-2-mercaptoimidazol, 2-Mercaptoimidazol, 2-Mercaptothiazol, 3-Mercapto-1,2,4-triazol, 5-Mercapto-1,2,3-triazol, 5-Mercapto-1,2,3,4-tetrazol und 2-Mercapto-1,3,4-thiadiazol ausgewählt ist; insbesondere wobei der Ligand N-Methyl-2-mercaptoimidazol, 2-Mercaptothiazol oder 2-Mercapto-1,3,4-thiadiazol ist.
  6. Pseudobioaffinitätschromatographie-Adsorptionsmittel nach Anspruch 4, wobei Y1 weder S, SCH3 + noch O ist.
  7. Pseüdobioaffinitätschromatographie-Adsorptionsmittel nach Anspruch 1 oder 4, wobei der Ligand auf der Oberfläche des festen Trägermaterials über ein bifunktionelles Aktivierungsmittel immobilisiert ist, das die Gruppe aufweist, die zu der Mercaptogruppe des Liganden reaktiv ist; insbesondere wobei das bifunktionelle Aktivierungsmittel aus Epichlorhydrin, Epibromhydrin, Dibrompropanol, Dichlorpropanol, Dibrombutan, Ethylenglykoldiglycidylether, Butandioldiglycidylether und Divinylsulfon ausgewählt ist.
  8. Pseudobioaffinitätschromatographie-Adsorptionsmittel nach einem der vorhergehenden Ansprüche, wobei der feste Träger ausgewählt ist aus Zellulose, Stärke, Dextran, Agar, Agarose, substituierten oder unsubstituierten Polyacrylamiden, Polymethacrylamiden, Polyacrylaten, Polymethacrylaten, Polyvinyl-hydrophilen Polymeren, Polystyrol und Polysulfon, Siliziumdioxid, Aluminiumoxid, Titandioxid, Zirkoniumdioxid, Polysaccharid-synthetischen Polymeren, Polysaccharid-Mineralstrukturen und synthetischen Polymer-Mineralstrukturen.
  9. Pseudobioaffinitätschromatographie-Adsorptionsmittel nach einem der vorhergehenden Ansprüche, wobei das feste Trägermaterial eine physikalische Form aufweist, die aus Kügelchen, unregelmäßigen Teilchen, Fasern, Membranen, flachen Oberflächen und schwammähnlichen Materialien ausgewählt ist.
  10. Verfahren zur Herstellung eines Pseudobioaffinitätschromatographie-Adsorptionsmittels zur Verwendung bei der Bindung eines Target-Proteins bzw. Zielproteins während Affinitätstrennungen des Target-Proteins von einem Gemisch von Verbindungen, wobei das Verfahren die folgenden Schritte umfasst:
    (a) Bereitstellen eines festen Trägermaterials;
    (b) Immobilisieren eines Liganden auf der Oberfläche des festen Trägermaterials über eine kovalente Bindung, die zwischen der Mercaptogruppe des Liganden und einer auf dem festen Trägermaterial vorliegenden reaktiven Gruppe ausgebildet wird, wobei der Ligand aus den Formeln I und II wie in einem der Ansprüche 1 bis 3 und 4 bis 6 definiert, ausgewählt wird, und wobei die reaktive Gruppe aus Epoxygruppen, Tosylaten, Tresylaten, Halogeniden und Vinylgruppen ausgewählt wird.
  11. Verfahren nach Anspruch 10, wobei der Immobilisierungsschritt das Aktivieren des festen Trägermaterials mit einem bifunktionellen Aktivierungsmittel, das die Gruppe aufweist, die zu der Mercaptogruppe des Liganden reaktiv ist, und das In-Kontakt-Bringen des Liganden mit dem aktivierten festen Trägermaterial umfasst; insbesondere wobei das bifunktionelle Aktivierungsmittel wie in Anspruch 7 definiert ist.
  12. Verfahren zur Durchführung von Affinitätstrennungen eines Target-Proteins bzw. Zielproteins, das von einem Gemisch von Verbindungen abzutrennen ist, wobei das Verfahren die folgenden Schritte umfasst:
    (a) Bereitstellen eines Pseudobioaffinitätschromatographie-Adsorptionsmittels wie in einem der Ansprüche 1 bis 3 und 4 bis 6 definiert;
    (b) In-Kontakt-Bringen des Gemisches von Verbindungen, das das Target-Protein enthält, mit dem Pseudobioaffinitätschromatographie-Adsorptionsmittel, wobei dem Gemisch von Verbindungen eine geeignete Menge eines Salzes hinzugefügt worden ist, um dem Target-Protein zu gestatten, an den Liganden zu binden; und
    (c) Aufrechterhalten eines Kontakts zwischen dem Gemisch und dem Pseudobioaffinitätschromatographie-Adsorptionsmittel für eine Zeit, die ausreichend ist, um das Target-Protein selektiv und reversibel an den immobilisierten Liganden zu binden, wodurch das Target-Protein von dem Gemisch abgetrennt wird.
  13. Verfahren nach Anspruch 12, wobei in dem Liganden von Formel I keines von X1, X2 und X3 S, SCH3 + oder O ist und die geeignete Menge an Salz Null beträgt ist.
  14. Verfahren nach Anspruch 12, wobei in dem Liganden von Formel II, Y1 weder S, SCH3 + noch O ist und die geeignete Menge an Salz Null beträgt.
  15. Verfahren nach einem der Ansprüche 12 bis 14, wobei das Target-Protein ein Immunoglobulin ist.
EP95919182A 1994-05-16 1995-05-16 Chromatographische adsorbenden, die merkaptoheterozyklische liganden anwenden Expired - Lifetime EP0764048B1 (de)

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